The invention relates to Chondrocytes and Chondrocyte Cell-Lines for use, particularly but not exclusively, for studying arthritic conditions.
In addition, the invention relates to chondrocytes which may be of particular advantage in the development of new biomaterials for use in replacing skeletal tissues such as hips and joints.
Arthritis is characterised by inflammation of the joints and thus an inflammation of cartilage tissue. Cartilage is made from materials secreted by cells, and specifically Chondrocytes, into extracellular space. Two of the most prominent materials to be secreted by cells are collagen and mucopolysaccharides. Mucopolysaccharides are large molecular weight sugar molecules comprising carbohydrate chains which exists as repeating disaccharides. One of the two sugars is always an amino sugar, either N-acetyl-glucosamine or N-acetyl-galactosamine. In most cases, mucopolysaccharides exist in combination with proteins and thus form proteoglycans.
Collagen is fibrous in structure whereas mucopolysaccharides are amorphous in appearance and extremely viscous and can thus occupy large volumes for a given weight of material. In many cases collagen and proteoglycans interact with one another to form a variety of extracellular structures. These structures appear to be self-organising; there is no evidence of enzymes or other materials which bring about their polymerisation, interaction or orientation. For example, a typical collagen proteoglycan found in cartilage may consist of a central filament of hyaluronic acid several micro meters long having bound thereto, at regular intervals, link proteins. Long core proteins bind to the link proteins and branching from the core proteins at regular intervals are mucopolysaccharides such as chondroitin sulphate and keratan sulphate. Thus extracellular matrix materials organise themselves to form cartilage.
Bone formation is characterised by calcification in the region of hypertrophic chondrocytes. Whilst vascular invasion is important for mineralisation in vivo, it is clear that initiation of calcification can be independent of vascularization, since hypertrophic chondrocytes can spontaneously mineralise their matrix in vitro in the absence of exogenous phosphate. However, adult articular cartilage does not normally calcify, except at its junction with subchondral bone where the calcification forms a characteristic tide-mark. It is therefore interesting to note that chondrocalcinosis, the pathological calcification of articular cartilage by calcium pyrophosphate, which occurs in different regions of articular and other cartilage, increases in prevalence with age and may be associated with osteoarthritis.
It therefore follows that understanding the mechanisms associated with osteoarthritis should involve an understanding of the mechanisms associated with calcification of articular cartilage. However, there are no cell models for this investigation. Rather, existing cell models employ the use of growth plate chondrocytes, that is to say chondrocytes which mineralise calcium in order to produce bone tissue. Using this chick cell model it has been possible to show that chick growth plate chondrocyte cell layers become mineralised in the presence of ascorbate or of beta-glycerophosphate (1). The effects of ascorbate are associated with an increase in the synthesis of alkaline phosphatase and type X collagen, though it is not clear if they are both essential for calcification to occur. Beta-glycerophosphate acts independently of regulation of these two molecules, presumably by providing a rich source of exogenous phosphate for nucleation. Other workers have developed a pellet culture system for rabbit growth plate chondrocytes in which there is synthesis of alkaline phosphatase and type X collagen as well as calcification in cultures with ascorbate (2). A separate study (3) suggests that the effects, of ascorbate may be species-specific, highlighting the need for a human chondrocyte model system if human disease processes are to be studied.
No studies to date have shown calcification of matrix in vitro by articular chondrocytes. Workers have compared rabbit articular and growth plate chondrocytes and found that in high-density cultures the growth plate chondrocytes produce alkaline phosphatase and incorporate calcium into the cell layers, whereas articular chondrocytes do not (4). However, two studies have demonstrated that chick (5) or human (6) articular chondrocytes can express type X collagen and alkaline phosphatase in long-term cultures, but, there is no evidence for calcification of cell layer matrix in either study.
Other workers have described in International Patent Application W09409118, the closest prior art, how a human chondrocyte cell-line can be produced by transfecting juvenile primary chondrocytes using an SV40 oncogene. The chondrocytes express Type II collagen a specific matter for articular chondrocytes. However, there is no reference to matrix calcification of matrix using these cell-lines.
It can therefore be seen that existing in vitro models of calcification rely on long-term cultures of animal growth plate chondrocytes. There is currently no model of human chondrocyte calcification.
We have surprisingly found that mature articular chondrocytes and immortalised articular chondrocyte cell-lines provide an extracellular matrix that rapidly calcifies. By the term mature, we mean chondrocytes from an elderly individual such as an individual of an age likely to development osteoarthritis such as, but not limited to, an individual over the age of 50, and more preferably 65. Or, alternatively, chondrocytes grown in vitro of functionally equivalent age.
Our finding therefore represents a valuable opportunity for the study of mechanisms associated with calcification of articular cartilage and also for the regulation of said calcification. We expect that our articular chondrocyte cell-lines will have both academic and commercial value in that they will not only facilitate a greater understanding of calcification that may be associated with the osteoarthritic condition but they will also serve as valuable tools for the development of therapeutic agents to treat such a condition.
We are unable to explain our findings but we speculate as follows. During development chondrocytes are typically committed to one of two lineages. The first concerns the production of bone and thus the differentiated chondrocytes mineralise calcium. The second lineage concerns the production of cartilage tissue and thus the differentiated chondrocytes do not mineralise calcium. It may be that there is some plasticity in the determination of lineage and, for example, this may be related to demethylation of DNA with ageing.
Thus as articular chondrocytes age the control mechanisms that prevent calcium mineralisation operate less effectively and surprisingly chondrocytes of the articular cartilage lineage actually mineralise calcium.
Our findings thus suggest that, contrary to previous expectations, it is possible to provide a human cell model for arthritis. The cell model can be provided using either mature or immortalised articular chondrocytes and ideally cell-lines pertaining thereto.
According to a first aspect of the invention there is therefore provided an articular chondrocyte which mineralises calcium.
Preferably there is provided a plurality of chondrocytes which are arranged in pellet form.
Preferably said chondrocyte and chondrocytes are derived from a mature individual, and ideally from such an individual suffering from or having a predisposition for arthritis. Alternatively, said chondrocyte and chondrocytes are of foetal origin and preferably human foetal origin. More preferably still said chondrocyte and chondrocytes are mature as herein defined.
Preferably further still there is provided cell-lines of such chondrocytes.
In preferred embodiments of the invention said cell-lines are of human origin and ideally comprise an oncogene and ideally a temperature sensitive oncogene so that at a first permissive temperature an active oncogene product is expressed and at a second non-permissive temperature expression of the active oncogene product is prevented. We have found that use of such an oncogene product provides for a cell-line which at the non-permissive temperature shows an increase in type II collagen expressionxe2x80x94a marker for the fully differentiated phenotype.
Ideally the oncogene is a temperature sensitive mutant of the immortalising oncogene SV40-T.
We are unsure, at this stage, whether calcium mineralisation by articular chondrocytes is directly responsible for osteoarthritis, or alternatively, whether calcium mineralisation by articular chondrocytes is an indicator of a yet unidentified condition or marker which is responsible for osteoarthritis. In either case, the cell-lines of our invention represent suitable cell models for further investigation.
As mentioned above, we are further unsure, at this stage, about the nature of the mechanisms which lead to calcium mineralisation by articular or hypertrophic chondrocytes. Other workers have suggested that there exists age related reactivation of genes (7). Thus as we speculated above it may be that a process of de-differentiation gives rise to osteoarthritis. It may be that genes which are typically inactive during life are switched on with ageing and are thus responsible for the aberrant phenotype of the mature articular chondrocyte.
The above speculation is provided for the purpose of edification only. It is not intended that the application should be limited by any of the above speculations, rather the speculations are provided for the purpose of comprehension.
There are obvious advantages to be derived from the use of in vitro cell models that mimic the arthritic condition. But, in addition we have produced chondrocyte cell-lines from foetal tissue which express type X collagen, a marker of hypertrophic chondrocytes. Such chondrocytes are known to mineralise calcium and therefore once the phenotype of these cells was determined the existence of mineralisation was less surprising. However, it is of note that no one has ever made a human hypertrophic chondrocyte cell-line before, despite its obvious advantages in understanding the mineralisation process symptomatic of the arthritis condition. Moreover, no one has thought to make such a cell-line for the study and/or production of joint matrix tissue for use in skeletal repair and/or replacement studies and/or surgery. We therefore also present herein knowledge of, and methods relating to, the production of a human hypertrophic chondrocyte cell-line.
According to a second aspect of the invention there is provided a human hypertrophic chondrocyte cell-line.
In a yet a further preferred embodiment of the invention said cell-line is of human foetal origin and also ideally comprises an oncogene and ideally a temperature sensitive oncogene so that at a first permissive temperature an active oncogene product is expressed and at a second non-permissive temperature expression of the active oncogene product is prevented. We have found that use of such an oncogene product provides for a cell-line which at the non-permissive temperature shows type X collagen expression, a marker for hypertrophic chondrocytes. Thus alternative cell-lines derived from human foetal tissue which mineralise calcium can be provided.
According to yet a further aspect of the invention there is provided a method for producing articular chondrocyte cell-lines, which method comprises:
a) immortalising at least one mature human articular chondrocyte cell using an immortalising agent; and
b) culturing said immortalised cell in order to produce a population of human articular chondrocyte cells.
Preferably the immortalising agent is an oncogene and more preferably still a temperature sensitive oncogene which at a non-permissive temperature results in enhanced expression of the phenotypic characteristics of the chondrocyte.
In a preferred embodiment of the invention immortalisation is achieved using conventional transfection techniques and preferably the immortalising agent is an immortalising gene such as an oncogene and more preferably still a temperature sensitive mutant of the immortalising oncogene SV40-T.
Ideally said method further involves culturing said immortalised cell at the non-permissive temperature of the oncogene.
According to a yet further aspect of the invention there is provided a method for producing hypertrophic chondrocyte cell-lines, which method comprises:
a) immortalising at least one human foetal chondrocyte cell using an immortalising agent; and
b) culturing said immortalised cell in order to produce a population of human foetal-derived chondrocyte cells.
Preferably the immortalising agent is an oncogene and more preferably still a temperature sensitive oncogene which at a non-permissive temperature results in enhanced expression of the phenotypic characteristics of the chondrocyte.
In a preferred embodiment of the invention immortalisation is achieved using conventional transfection techniques and preferably the immortalising agent is an immortalising gene such as an oncogene and more preferably still a temperature sensitive mutant of the immortalising oncogene SV40-T. More preferably still said transfection is undertaken using retroviral temperature sensitive oncogene transduction.
Ideally said method further involves culturing said immortalised cell at the non-permissive temperature of the oncogene.
According to a yet further aspect of the invention there is provided chondrocytes, or at least one chondrocyte cell-line, in accordance with the invention for use in developing therapeutic agents designed to treat arthritis.
According to a yet further aspect of the invention there is provided a method for developing therapeutic agents designed to treat arthritis comprising:
a) exposing chondrocytes, or at least one chondrocyte cell-line, in accordance with the invention to at least one test agent; and
b) observing the nature of the response to said test agent.
In a preferred method of the invention said observation involves an assessment of the amount of calcium mineralisation associated with said chondrocytes or said chondrocyte cell-lines.
According to an alternative aspect of the invention there is provided hypertrophic chondrocytes, or at least one hypertrophic chondrocyte cell-line, for use in providing joint matrix tissue.
According to an alternative aspect of the invention there is provided a method of producing joint matrix tissue for use in repair and/or replacement of damaged tissue comprising:
a) providing a hypertrophic chondrocyte cell-line;
b) culturing said cell-line under conditions that promote matrix growth; and
c) harvesting said cells and/or said matrix tissue and/or selected parts thereof.